Dexamethasone abolishes the activation by nerve growth factor of protein kinase N: effects of nerve growth factor and dexamethasone on protein kinase N

Neuroscience Letters, 159 (1993) 119-122 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940•93•5

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Dexamethasone abolishes the activation by nerve growth factor of protein kinase N: effects of nerve growth factor and dexamethasone on protein kinase N Cinzia Volont6 Department of Pathology and Centerfor Neurobiologyand Behavior, College of Physicians and Surgeons of Columbia University, New York, N Y 10032 (USA) (Received 20 January 1993; Revised version received 10 May 1993; Accepted 11 May 1993)

Key words." Nerve growth factor; Dexamethasone; PC12 cell; Protein kinase N Protein kinase N (PKN) is a basic 4547 kDa serine/threonine protein kinase activated by NGF and several other factors in PCl2 cells and other cell types. It is inhibited in vitro by purine analogs. In this work, we further characterize the modulation of PKN activity following exposure of PC 12 cells to dexamethasone or NGF, which respectively direct these cells towards the chromaffln- or neuron-like phenotype. We show here that the two factors elicit opposite effects on the activation of the kinase: dexamethasone inhibits while NGF stimulates the basal level of PKN activity. Simultaneous addition to the cells of the factors causes no variation from basal kinase activity. Addition of dexamethasone not to the cells, but to partially purified PKN during the kinase assay, causes no modulation of the enzyme.

Glucocorticoid hormones act in nearly all cell types of eukaryotic organism [1, 14, 16] as important regulators of normal development, growth and differentiation [9, 13, 20]. They exert biological effects by binding to specific intracellular soluble receptors [30], thereby triggering a cascade of events that culminates with the interaction of the receptor with specific sites on the DNA and with the modulation of transcription of target genes [15, 17,311. The PC12 pheochromocytoma cell line [7] is a useful cellular model system for studying how the dynamism of the microenvironment dictates the choice of expression of a particular set of genes and therefore how extracellular signals can modulate cellular plasticity. In the specific case, glucocorticoids direct PC12 cells towards a chromaffin-like phenotype [2-5, 12, 21], while NGF [11] induces differentiation of the cells into sympathetic neurons [10]. Protein kinase N (PKN) [18, 27] is a basic 45-47 KDa serine/threonine protein kinase activated by NGF and other factors in PC12 cells [19] and in several non-neuronal cell lines that possess functional NGF receptors

Correspondence: C. Volont6, Institute of Neurobiology, CNR, Viale Marx 15, 00137 Rome, Italy. Fax: (39) (6) 822203.

[23]. In vitro, PKN uses histone HF1, tyrosine hydroxylase, synapsin, ribosomal $6 protein, c-fos, c-jun and myelin basic proteins as substrates; it is soluble, it is not inhibited by Mn 2+ and it does not require Ca 2÷, cAMP or other cofactors. PKN is inhibited in vitro by purine analogs [24], some of which are potent and possibly specific [26] inhibitors of this kinase, and is apparently involved in certain of the multiple pathways of the NGF mechanism of action [8, 24-26]. A PKN-like activity is furthermore associated with both Trk and p75 NGF receptors [28, 29]. In the present work we investigate how the activity of PKN is modulated after exposure of PCl2 cells to distinct differentiating agents, such as dexamethasone or NGF. Extraction of soluble cellular protein and elution of PKN from Fast S mini-column with 300 mM NaCl in extraction buffer (50 mM HEPES, pH 7.4, 2 mM EGTA, 10 mM NaF, 10 mM MnCl> 2 mM PMSF, 100 KIU/ml Trasylol) were carried out as described [23]. Aliquots of PKN-enriched fractions (30-40/.d) were then mixed with 10/~g of histone HF1, and phosphorylation assays were started by the addition of 1/.tCi of [7-32p]ATP (3000 Ci/ mmol, NEN). This mixture was incubated for 10 min at 37°C, and the phosphorylation reactions were terminated by the addition of 4x-concentrated electrophoretic sample buffer and filtration of the samples

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under vacuum onto nitrocellulose, in order to separate the phosphorylated substrate from free [7-32p]ATE all as described [23]. We show here that culturing PCI2 cells for 5 min with N G F causes a rapid 2-fold activation of PKN (Fig. 1). The addition to the cells of 1 ,uM dexamethasone inhibits instead in 5 min about 65% and in 30 min 80- 100% of PKN basal activity (Figs. 1 and 3). After the simultaneous addition of N G F and dexamethasone (30 min), the activity of PKN is not very different from the basal level generally detected in untreated PC12 cells (Fig. 1), thereby suggesting that the two agents offset their respective biological actions on PKN. When dexamethasone is added to partially purified PKN during the in vitro assay of the kinase, the compound does not interfere with either basal or NGF-induced kinase activity (Fig. 2). This indicates that the mechanism responsible for the inhibition of PKN in intact cells could be either a direct effect of dexamethasone on the enzyme (or enzymes) modulating both the basal and the NGF-activated P K N activity, or simply an indirect uncoupling of the reactions that maintain or stimulate (following N G F treatment) the activity of PKN. The data presented here also indicate that dexamethasone can exert a very rapid and probably transcription-independent biological effect on target cells. A dose-response experiment shows that dexamethasone inhibits the NGF-induced activation of PKN with IC~0 in

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Fig. 1. Time-dependent bimodal regulation of P K N activity. PC12 cells were cultured in the presence of N G F (50 ng/ml) or dexamethasone ( I ,uM) for different times. P K N was then partially purified from cell extract (750 p g of total protein) as described in ref. 23. The kinase was tested using histone HF1 (10/1g/assay) as a substrate, in a final volume of 65/11. The phosphorylation assay was performed for 10 min at 37°C in the presence of 1 pCi of [7-32p]ATP (11 ,uM final concentration). Specifically incorporated radioactivity was measured as described in ref. 23. Data are shown as means of triplicate determinations _+ S.E.M. Comparable results were obtained in three independent experiments.

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(nM) Dexamethasone in vitro Fig. 2. Dexamethasone does not inhibit P K N in vitro. PC12 cells were cultured in the absence or presence of N G F (50 ng/ml). P K N was then partially purified from cell extract (750/lg of total protein) as described in ref. 23. The kinase was tested in vitro in the absence or in the presence of different concentrations of dexamethasone, using histone HFI ( 10/~g/assay) as a substrate, in a final volume of 65 ,ul. The phosphorylation assay was performed for 10 min at 37°C in the presence of 1 ,uCi of [7-32p]ATP (11 ,uM final concentration). Specifically incorporated radioactivity was measured as described in ref. 23~ Data are shown as means of triplicate determinations +_ S.E.M. Comparable results were obtained in two independent experiments.

the 100-150 nM range. The basal level of PKN activity is instead inhibited by dexamethasone with IC50 in the 10 15 nM range (Fig. 3). This difference in ICs0 suggests that two different enzymes or two different post-translational modifications of the same enzyme could modulate the basal and the NGF-activated PKN. It was previously shown that dexamethasone decreases the amount of N G F receptors in PC12 cells [22]. In the present case, N G F receptor downregulation is unlikely to be responsible for inhibition of P K N activation, due to the very short exposure of the cells to the compound. It was also recently reported that, similarly to PKN modulation, GAP-43 expression is inhibited by dexamethasone but stimulated by N G F [6]. Our data therefore support a bimodal regulation of biological effects in PCI2 cells by these compounds. Irrespective of whether N G F and dexamethasone stimulate or inhibit the activity of P K N in PC12 cells, our findings in turn raise the issue of the biological significance of such a bimodal effect. One possibility from these and previous results is that NGF-dependent activation of PKN could participate in the induction of the neural phenotype of PC12 cells. Similarly, inhibition of the kinase by dexamethasone could facilitate the chromaffin-

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(nM) Dexamethasone in intact cells Fig. 3. Dose-response for dexamethasone-dependent inhibition of PKN activity. PC12 cells were cultured for 30 min in the absence or presence of NGF (50 ng/ml)+ different concentrations of dexamethasone. PKN was then partially purified from cell extract (400 fig of total protein) as described in ref. 23. The kinase was tested using histone HF1 (10 fig/assay) as a substrate, in a final volume of 65 pl. The phosphorylation assay was performed for 10 min at 37°C in the presence of 1 /aCi of [~'-32p]ATP (11 /aM final concentration). Specifically incorporated radioactivity was measured as described in ref. 23. Data are shown as means of triplicate determinations + S.E.M. Comparable results were obtained in three independent experiments.

like state. We can find support for this hypothesis both in previous work which shows that dexamethasone impairs the NGF-induced fiber outgrowth from adrenal chromaffin cells and in our observation that 6-thioguanine and 6-methylmercaptopurine riboside (two potent inhibitors of PKN) prevent the NGF-dependent regeneration of neurites from PC12 cells [8, 24]. The author would like to thank Dr. L.A. Greene for useful discussions. The technical assistance of F. Negri

wasgreatlyappreciated.

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